• Non ci sono risultati.

MECHANISMS OF INTERACTION WITH INHIBITORS

Introduction and aim of the work

Fluctuations in the brain levels of the neuromodulator KYNA may control cognitive process and play a role in several neurological brain diseases (1). Since KAT II accounts for the majority of KYNA formation in the human brain, a KAT II selective inhibitor is an important pharmacological tool to control KYNA levels favouring precognitive effects. The potential harmful complete depletion of this neuroprotective molecule would be prevented by the action of other KAT isoenzymes (2).

Development of isozyme-specific inhibitors is notoriously difficult, especially in the case of PLP-dependent aminotransferases, since the members of this family share significant conservation of the active site (3). To this end, the different geometry of the ligand-binding pocket in KAT I and II isozymes was exploited for the synthesis of the first potent and selective rat KAT II inhibitor, (S)-4-ethylsulfonylbenzoylalanine (S-ESBA), a close structural analogue of KYN that bears a bulky substituent on the anthranilic ring (4). ESBA was found to be pharmacologically active on rats KAT II but poorly active towards the human ortholog, as signaled by a 20-fold higher IC50 value (4, 5).

On the basis of the results of a molecular docking approach, it was proposed that this different inhibitory activity might arise from the presence in the catalytic site of human KAT II of two hydrophobic residues, Leu40 and Pro76, which are substituted by polar serine residues in the rat enzyme (5). Recently, a human KAT II double-mutant harboring the serine residues characterizing the rat ortholog active site was generated, in order to investigate the molecular basis for ESBA species-specificity (2). Unexpectedly, the site-directed mutagenesis approach did not provide further experimental support to explain the striking difference in ESBA inhibitory efficiencies toward rat and human KAT II, and underlined the need for more in depth biochemical investigation aimed at deciphering the mechanism of ESBA inhibition. It is also worth noticing that rat KAT II displays a KM for KYN of 0.66 mM (6), a value about ten-fold lower than the value determined for hKAT II (7).

This observation indicates that the rat enzyme has evolved to recognize the physiological substrate KYN with a higher affinity compared with the human KAT II. Interestingly, both KYN and ESBA bind more tightly to the active site of the rat KAT II, suggesting that their binding is controlled by a similar mechanism. In this respect, it can be noted that S-ESBA retains the general amino acid chemical structure, opening the question whether this molecule is actually more a substrate than an inhibitor in the case of the human enzyme (2). This hypothesis can be investigated by determining the mechanism of S-ESBA action on hKAT II.

The isotype-specificity of a rat KAT II inhibitor, cysteine sulphinate (CSA), has been demonstrated for brain slices (8) and might be the starting point for the development of potent and specific inhibitors of the synthesis of KYNA in the brain. In mammals, endogenous sulphur-containing

amino acids, such as homocysteine, cysteine sulphinate, homocysteine sulphinate and cysteate, display neuroexcitatory actions similar to those of glutamate. These cysteine derivatives were able to reduce the production of KYNA in cortical slices in rats, due to their interaction with KATs. Thus, they were considered endogenous modulator of KYNA synthesis in the CNS (9, 10). In particular, CSA displayed unique specificity, inhibiting only the action of rat KAT II and showing an IC50 of approximately 2 M (8), relatively close to the physiological brain level of CSA (11, 12). The potency of CSA as an inhibitor of human KAT II has never been evaluated so far. Moreover, the mechanisms of both CSA and ESBA inhibition on human KAT II have not yet been studied in depth. Therefore, a detailed analysis of the mechanisms of action of these two inhibitors was carried out.

CSA ESBA

Materials and Methods

Materials. L-Kynurenine (K8625), CSA (C4418), glutamate oxidase from Streptomyces sp.

(G5921), peroxidase from horseradish (P8375), o-dianisidine (D9143) were purchased from Sigma-Aldrich (St.Louis, MO). ESBA was synthesized as previously described (4, 5) and kindly provided by Prof. Roberto Pellicciari.

KAT transamination activity measured by a coupled glutamate oxidase-peroxidase assay. The amount of glutamate produced by transamination activity in the presence of KG was determined by an end-point assay based on the use of GOX and peroxidase. A reaction mixture containing 10 mM KG, 40 M PLP and the potential aminogroup donor (CSA or ESBA), was equilibrated at the desired reaction temperature (25°C) in 50 mM Hepes, pH 7.5, before starting the reaction with addition of 2 M KAT II. Aliquots (20 L each) were removed after an appropriate incubation time, and stopped by mixing with 2 L of 1.14 M phosphoric acid (final concentration 14 mM). Each aliquot was subsequently mixed with a detection solution containing 0.02 units of glutamate oxidase (GOX-Sigma G5921), 3 units of peroxidase (perox-Sigma P8375), 1 mM O-dianisidine (Sigma D9143) and 50 mM Hepes pH 7.5, in a final volume of 200 L. The detection mixture was incubated at 37 °C for 30 minutes. Finally, the mixture was supplemented with 50 L sulphuric acid

(final concentration 3.36 mM) to dissolve the occasional precipitates of oxidized O-dianisidine and to increase the sensitivity of the assay (13), before measuring the absorbance at 530 nm to quantify the extent of O-dianisidine oxidation. A calibration curve was prepared, using known concentrations of glutamate, ranging from 10 to 800 M in the presence of 10 mM KG, 40 M PLP, 14 mM phosphoric acid, 1 mM O-dianisidine (Sigma D9143), 0.02 units of glutamate oxidase (GOX-Sigma G5921), 3 units of peroxidase (Perox-Sigma P8375) and 50 mM Hepes pH 7.5, in a final volume of 200 L. The reaction mixture was incubated and treated as above, and the absorbance at 530 nm was plotted against the initial amount of glutamate to generate a calibration curve. Blanks (control reactions) were set up using the same reagents as for the assay except for hKAT II that was replaced by the same volume of buffer.

KAT -elimination activity measured by Nessler’s assay. Reaction mixtures containing ammonia produced by -elimination were tested with the Nessler’s assay (14) using a ready-to-use solution (Fluka, code 72190). At pH 7.5 98% of ammonia is in the NH4+ form and the solubility of NH3 in water at 25 °C is about 50 % (w/w), thus no significant loss of ammonia by evaporation is expected under these experimental conditions. A standard curve was built using ammonium sulfate solutions at concentrations in the range 1-20 mM. 40 l of a solution containing ammonia were diluted with 860 l of water. 100 l of Nessler’s reagent were added to the mixture and the absorbance of the solution immediately recorded at 436 nm.

Determination of CSA inhibition mechanism and dissociation constants. The mechanism of inhibition of CSA and its dissociation constants were determined by measuring the initial rate of reactions containing 870 nM hKAT II in 50 Mm Hepes pH 7.5 in the presence of 10 mM KG, 40 M PLP and concentrations of KYN from 2.5 to 10 mM. The reactions were carried out at 25 °C in 0.1 cm optical path cuvettes either in the absence or presence of 4 mM and 20 mM CSA. Initial rates were measured from kinetic traces collected at 310 nm using the calculated Δ 310 nm = 3625 M-1 cm-1 for KYNA.

Double reciprocal plots for uncompetitive inhibition of KAT II by CSA were globally fitted to linear equations of the type (15):

ii

0 K

1 CSA appV

1 KYN

1 appV

appK V

1 [1]

where appK and appV are apparent KM and apparent Vmax. The slope is the linking parameter, whereas the intercept is allowed to vary. A secondary plot of intercepts of the primary plots versus [CSA] gives an estimated Kii as the abscissa intercept. Because the KYN concentration used in the assay is close to KM, an approximated value of Ki can be obtained by the following:

KG M ii i

K 1 KG 2

K K [2]

Determination of kinetics parameters for transamination reaction with ESBA. A continuous assay for the determination of kinetic parameters for the transamination reaction of ESBA with hKAT II was developed (see Results). Reactions (200 L final volume) containing 50 mM Hepes pH 7.5, 10 mM KG, 40 M PLP, and variable concentrations of ESBA (50 M-15 mM) were incubated at 25°C. The reaction was started by the addition of 870 nM hKAT II and carried out at 25 °C in a 0.1 cm optical path cuvette. Initial rates were estimated exploiting the absorbance increase at 338 nm due to the accumulation of the ketoacid ESdiOBA ( = 15,400 M-1∙cm-1).

Results

Reactivity with CSA.

CSA is a physiological substrate of AspAT (16, 17) due to its structural similarity with Asp. On the basis of structural considerations, CSA can, in principle, be a substrate for either transamination and -elimination reaction catalyzed by KAT II, being sulfite a good leaving group. The reaction of hKAT II with CSA was first analyzed spectroscopically (Figure 1A). hKAT II reacts with 1.8 mM CSA showing a slow decrease of the intensity of the internal aldimine band at 360 nm and the accumulation of a species, likely PMP, absorbing at 330 nm. The reaction is completed in about 60 minutes. In the presence of the same enzyme concentration and 10 mM -aminoadipate the reaction reaches equilibrium within 8 minutes (see Figure 1C in chapter 2). The occurrence of transamination activity on CSA in the presence of KG was demonstrated by a GOX-peroxidase coupled assay measuring the formation of glutamate. For the same reaction, the accumulation of ammonia was detected by a Nessler’s assay, indicating that also a -lytic side reaction takes place. Cysteine sulfinate is thus a poor substrate of KAT II rather than a pure, competitive inhibitor.

To further investigate CSA mechanism of action, we exploited the new developed continuous spectrophometric assay for KYN aminotransferase activity monitoring the absorbance increase at 310 nm. hKAT II activity assays were carried out at two concentrations of cysteine sulfinate, namely 4 and 20 mM. A double reciprocal plot of the initial velocity against KYN concentration gave a series of parallel lines, indicative of an uncompetitive inhibition (Figure 1B). Double reciprocal plots for uncompetitive inhibition of hKAT II by CSA were globally fitted to equation 1 with a slope of 392±22 min and intercepts of 47±4, 68±6 and 94±6 mM-1 in the presence of 0, 4 and 20 mM CSA, respectively. A secondary plot of the intercepts of the first plot versus inhibitor concentration gave a straight line, allowing to estimate a K for CSA of about 25 mM (Figure 1B,

inset) (15). The approximated value of Ki, calculated from equation 2, is 20 M, in good agreement with the IC50 value from in vivo experiments on rats (8), which is about 2 M. These experiments provide the first information on the mechanism of inhibition of hKAT II by CSA.

Figure 1. Reaction of hKAT II with CSA

A Reaction of KATIIwith CSA monitored by absorption spectroscopy.

Spectra of the reaction mixture containing 7 M hKAT II in 50 mM Hepes, pH 7.5 at 25°C, were recorded in the absence (solid line) and presence of 1.8 mM CSA upon 5 (dotted line), 10 (short dashed line), 15 (dash dotted line) and 60 (long dashed line) minutes from the addition. Inset: time course of internal aldimine disappearance at 360 nm. The solid line represents the fitting to a monoexponential equation with k = 0.09 min-1.

1/[KYN] (1/mM)

-0,4 -0,2 0,0 0,2 0,4

1/V0 (min/mM)

0 75 150 225 300 375

[CSA] (mM) -30 -20 -10 0 10 20 30

Intercept

0 40 80 120

Wavelength (nm)

300 350 400 450 500 550 600

Absorbance

0,00 0,02 0,04 0,06 0,08 0,10

Time (min)

0 20 40 60

Absorbance 360 nm

0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07

A

B

B Determination of the mechanism of inhibition and inhibition constants.

The primary double reciprocal plot for KAT II inhibition by CSA was obtained by determining the rate of reaction in a mixture containing 870 nM hKAT II in 50 Mm Hepes pH 7.5 in the presence of 10 mM KG, 40 M PLP and concentrations of KYN from 2.5 to 10 mM. The reaction was carried out at 25 °C in 0.1 cm optical path cuvettes either in the absence (closed circles) or presence of 4 mM (open squares) and 20 mM CSA (open triangles). The solid lines through data points represent a global fitting to equation 1 with a slope of 392±22 min and intercepts of 47±4, 68±6 and 94±6 mM-1 in the presence of 0, 4 and 20 mM CSA, respectively. Inset: secondary plot of the intercepts of the primary plot versus CSA concentration. The abscissa intercept, 25 mM, gives an estimate of the inhibition constant Kii.

Reactivity with S-ESBA

ESBA is an aromatic compound analogous to the natural substrate KYN, that absorbs maximally at 287 nm, with an approximate extinction coefficient of 2050 M-1 cm-1 (Figure 2A, short dashed line).

ESBA might be either a pure inhibitor, as previously proposed (4), or, more likely, a substrate analog. Indeed, the structure of ESBA bearing an amino acid moiety suggests that this molecule might be processed by the enzyme.

The interaction between ESBA and hKAT II was first investigated by analyzing the spectroscopic behaviour of the enzyme in its presence. Binding of ESBA to hKAT II, in the absence of 2-oxoacids, led to marked changes in the absorption spectrum, with intensity increase at 283 nm and at around 330 nm. (Figure 2A). The absorption spectrum of the reaction mixture changes with time up to 11’ (dashed dotted line), indicating that ESBA is processed by the enzyme. Moreover, the final spectrum is similar but not coincident with the spectrum of PMP-KAT II, as collected in the presence of AAD (grey solid line). In particular, considering that in the absence of keto acids only one transamination cycle can take place, with consumption of an amount of ESBA equal to the amount of active sites, the product of the reaction show higher absorbance intensities at around 300 and 355 nm. The addition to the reaction mixture of 10 mM KG led to the accumulation of a species absorbing maximally at 338 nm (Figure 2B), likely the product of ESBA transamination, 4-(4-(ethylsulfonyl)phenyl)-2,4-dioxobutanoic acid (ESdiOBA) (Scheme 1). Spectral analysis of the product, after separation by ultrafiltration from the enzyme, showed a species with a single absorption peak centered at 338 nm (data not shown). As most keto acids significantly absorb in the 320-340 nm region (18), it seems likely, from its spectroscopic properties, that this product represents the keto acid generated by ESBA transamination. As a check, the occurance of transamination activity on ESBA was assessed by a GOX-peroxidase coupled assay for the same reaction mixture described in figure 2. The amount of ESBA transaminated by hKAT II at equilibrium (about 3 hours after the addition of KG) was found to be about the 90 % of the initial

ESBA concentration. Thus, the main product of the reaction is ESdiOBA, that is characterized by an extinction coefficient at 338 nm of 15,400 M-1∙cm-1.

Wavelength (nm)

300 350 400 450 500 550

Absorbance

0,0 0,5 1,0 1,5 2,0

Wavelength (nm)

300 350 400 450 500 550

Absorbance

0,0 0,2 0,4 0,6 0,8

A

B

Figure 2. Reactivity of KATII towards ESBA

A Absorption spectra recorded for a solution containing 8 M hKAT II in 50 mM Hepes, pH 7.5, 25

°C (black solid line), and in the presence of 100 M ESBA at the equilibrium (dashed dotted line).

For comparison, a spectrum of a solution containing 100 M ESBA in 50 mM Hepes, pH 7.5 (short dashed line), and the spectrum of PMP-KAT II collected in the presence of AAD (grey solid line) are reported.

B Upon addition of 10 mM KG a series of spectra was collected after 1’ (long dashed line) , 5’

(short dashed line), 15’ (medium dashed), 45’ (dotted line) and 185’ (grey dashed dot-dot line).

Spectra were corrected for KG contribution.

Based on previous experiments carried out in the presence of compounds undergoing -elimination, it was verify whether a -lytic reaction takes place on ESBA. In fact, it is reported in the literature that p-substituted benzaldehyde is produced by the elimination activity of kynureninase on kynurenine (19). If a -elimination reaction on ESBA is taking place, accumulation of ammonia is expected and can be compared to the amount of ammonia produced in the reaction with the good -elimination substrate BCA. The rate of -elimination was determined by monitoring the formation of ammonia as a function of time for a solution containing 2 M KAT II, 8 mM ESBA and 10 mM KG (Figure 3). The reaction is linear within 180’, with a slope of 2.5 M/min ammonia (e.g. the specific activity is 25 pmol/ g∙min). This rate is expected to be a lower limit, since, for substrates with poor leaving groups, transamination reaction, in the presence of 2-oxo acids, is favoured with respect to the -elimination reaction. As a comparison, the reaction of KAT II with 5 mM BCA gave a specific activity of 5 nmol/ g∙min, thus indicating that ESBA is a poor substrate for

-elimination.

Figure 3. -elimination kinetics of ESBA by hKAT II

The amount of ammonia formed in a reaction mixture containing 2 M hKAT II, 8 mM ESBA and 10 mM KG was determined as a function of time with the Nessler’s assay. The solid line through data points represents the fitting to a linear equation with slope 2.5 0.18 M/min.

Time (minutes)

0 50 100 150 200

[NH4+ ] (M)

0 100 200 300 400 500 600

Taken together these findings confirm that, as previously supposed, ESBA is a good substrate for hKAT II rather than a pure competitive inhibitor, and indicate two possible pathways for ESBA processing by hKAT II, as shown in Scheme 1. The reaction of hKAT II with ESBA proceeds mainly via a transamination reaction, producing 4-(4-(ethylsulfonyl)phenyl)-2,4-dioxobutanoic acid (ESdiOBA). This reaction is accompanied by a -elimination side-reaction that likely gives a p-substituted benzaldehyde. Both products are expected to absorb at wavelengths lower than 350 nm (18, 20) and are merely hypothesized, as any attemps to identify them by mass spectrometry failed.

Scheme 1. Reaction pathways for ESBA (in the presence of KG as amino group acceptor) to give either 4-(4-(ethylsulfonyl)phenyl)-2,4-dioxobutanoic acid (ESdiOBA) or 4-(ethylsulfonyl)benzaldehyde.

We have also assessed whether ESBA or its reaction products inactivate hKAT II, as it was observed with BCA. A solution of KAT II (174 M) was incubated with 8 mM ESBA for 60 minutes, at 25 °C. The reaction was diluted 200 fold in an assay solution containing 10 mM KYN and 10 mM KG. hKAT II reacted with ESBA was found to be two fold less active than the unreacted enzyme, suggesting that -lytic activity of ESBA might lead to syncatalytic inactivation of the enzyme. The enzyme that reacted with ESBA was unstable, as a PLP-ESBA derivative could not be recovered

neither after ultrafiltration nor gel filtration on micro spin columns. This hampered the determination of the type of modification by mass spectrometry.

Unlike in the case of CSA, the mechanism of inhibition of ESBA on hKAT II could not be determined, due to the heavy interference of ESBA spectrum with spectroscopic signals used to monitor hKAT II activity. However, the affinity of ESBA for hKAT II could be estimated exploiting the absorbance increase at 338 nm due to the accumulation of the ketoacid ESdiOBA. Kinetic parameters for the transamination reaction of ESBA with hKAT II were determined by collecting kinetic traces at 338 nm at different ESBA concentrations. Data were fitted to the Michaelis Menten equation with KM = 4.5 ± 0.9 mM and Vmax = 7.8 ± 0.6 M/min (Figure 4). kcat for the reaction of hKAT II with ESBA is 9 min-1, only about 2.5 fold less than the value of 25 min-1 for the reaction with KYN. Moreover, the inhibition of hKAT II by ESBA was further assessed by a colorimetric hightroughput screening assay, as illustrated in the following chapter.

Figure 4. Dependence of the rate of reaction of hKATnII on ESBA concentration in the presence of KG. The reaction mixture contained 870 nM hKAT II in 50 mM Hepes pH 7.5 in the presence of 10 mM KG and variable concentrations of ESBA (50 M-15 mM). The reaction was carried out at 25 °C in 0.1 cm optical path cuvettes. The solid line through data points represents the fitting to the Michaelis Menten equation with Vmax = 7.8 ± 0.6 M/min and KM = 4.5 ± 0.9 mM.

Discussion

In vivo experiments have indicated that both CSA and ESBA are inhibitors of KAT II (4, 5, 8), but their mechanisms of action were never studied in depth. On the basis of structure-reactivity considerations, they might be substrates for either transamination and -elimination reactions.

[ESBA] (mM)

0 4 8 12 16

V0 (M/min)

0 1 2 3 4 5 6 7

Their interactions with hKAT II were evaluated by both spectroscopic analysis and monitoring the accumulation of reaction products. Our findings demonstrate that both CSA and ESBA are substrate analogs and not purely competitive inhibitors.

CSA is a physiological substrate of AspAT that is able to catalyze both the transamination (21, 22) of CSA and its -elimination as a side reaction (23). CSA is an effective amino donor substrate for AspAT, probably because a product of the aminotransferase reaction, -sulfinylpyruvate, decomposes non enzymatically to SO32- and pyruvate, and hence the aminotransferase reaction is irreversible (21). In the case of hKAT II, cysteine sulfinate is a very slow transamination substrate when compared to the natural substrate AAD. Inhibition of hKAT II by CSA was determined to be uncompetitive, a quite unexpected finding, considering that CSA is also a substrate of hKAT II, converting the PLP form of the enzyme to the PMP form. Uncompetitive inhibition involves the exclusive (or predominant) binding of the inhibitor to the enzyme-substrate complex or to any intermediate downstream of it (15). This mode of inhibition is quite rare in nature and is particularly encountered in multi-substrate reactions (24-29), where the inhibitor is competitive with respect to one substrate (S2) but not with respect to another (S1). The reaction scheme is represented by

Inhibition occurs since ES1I is catalytically inactive. As it can not form product, it is a dead end complex which has only one fate, to returns ES1. The uncompetitive inhibition is most noticeable at high substrate concentrations (i.e. S1 in the scheme above) and cannot be overcome as both the Vmax and KM are equally reduced. Normally, the uncompetitive inhibitor also bears some structural similarity to one of the substrates. In the case of CSA, uncompetitive inhibition could come from preferential binding of CSA to the PMP form of hKAT II, suggesting that CSA might better mimic KG than KYN or AAD.

In the case of ESBA, the mechanism of inhibition could not be determined due to its strong interference with the continuous spectrophotometric assay for KYN aminotransferse activity.

However, the spectroscopic signal generated by the accumulation of the product of the reaction of ESBA with hKAT II was exploited to calculate the kinetic parameters for the transamination of ESBA by hKAT II and, thus, an approximate affinity of the inhibitor for the enzyme. An apparent KM

of 4.5 mM is in good agreement with a 64 % inhibition exerted by 1 mM ESBA (5). Any attempt to

identify the reaction products by mass spectrometry was unsuccessful. Although the identity of the product could not be assessed, it seems likely, from its spectroscopic properties, that it represents the -ketoacid generated by ESBA transamination. Occurrence of transamination activity on ESBA was confirmed by the detection of the other product of transamination activity, glutamate. As expected on the basis of ESBA structure, this substrate analogue is processed by hKAT II mainly via a transamination reaction, which is accompanied by a -lyase side activity. A rough estimate of the catalytic efficiency for -elimination reaction, based on specific activity at a fixed substrate concentration, indicate that ESBA, as KYN, is a poor substrate for -elimination, when compared to BCA. However, ESBA, like BCA, is capable of permanently inactivating hKAT II through a mechanism that, likely, involves formation of a covalent adduct between the active site lysine residue and the –aminoacrylate intermediate, as already reported for alanine aminotransferase (30) and AspAT (31) (see Scheme 4 in chapter 3).

In conclusion, although both CSA and ESBA show good inhibitory properties on hKAT II, with, at least for CSA, inhibition constants in the micromolar range, these molecules are actually substrates. In particular, the nature of the reaction of ESBA with hKAT II, possibly involving a syncatalytic inactivation and a -elimination reaction with production of an aromatic aldehyde, suggest caution in the clinical application of this molecule or its structural analogues.

Understanding the mechanisms of hKAT II inhibitors is important not only to enable the design of better hKAT II inhibitors, but also to understand the enzyme reaction mechanisms. Control of reaction mechanism is particularly important for PLP-dependent enzymes because it is known that they catalyze side reactions at significant rates with substrates analogues or even with their natural substrates (3).

REFERENCES

1. Rossi, F., Valentina, C., Garavaglia, S., Sathyasaikumar, K. V., Schwarcz, R., Kojima, S., Okuwaki, K., Ono, S., Kajii, Y., and Rizzi, M. Crystal structure-based selective targeting of the pyridoxal 5'-phosphate dependent enzyme kynurenine aminotransferase II for cognitive enhancement, Journal of medicinal chemistry 53, 5684-5689.

2. Casazza, V., Rossi, F., and Rizzi, M. Biochemical and Structural Investigations on Kynurenine Aminotransferase II: An Example of Conformation-Driven Species-Specific Inhibition?, Current topics in medicinal chemistry.

3. Amadasi, A., Bertoldi, M., Contestabile, R., Bettati, S., Cellini, B., di Salvo, M. L., Borri-Voltattorni, C., Bossa, F., and Mozzarelli, A. (2007) Pyridoxal 5'-phosphate enzymes as targets for therapeutic agents, Current medicinal chemistry 14, 1291-1324.

4. Pellicciari, R., Rizzo, R. C., Costantino, G., Marinozzi, M., Amori, L., Guidetti, P., Wu, H. Q., and Schwarcz, R. (2006) Modulators of the kynurenine pathway of tryptophan metabolism:

synthesis and preliminary biological evaluation of (S)-4-(ethylsulfonyl)benzoylalanine, a potent and selective kynurenine aminotransferase II (KAT II) inhibitor, ChemMedChem 1, 528-531.

5. Pellicciari, R., Venturoni, F., Bellocchi, D., Carotti, A., Marinozzi, M., Macchiarulo, A., Amori, L., and Schwarcz, R. (2008) Sequence variants in kynurenine aminotransferase II (KAT II) orthologs determine different potencies of the inhibitor S-ESBA, ChemMedChem 3, 1199-1202.

6. Guidetti, P., Amori, L., Sapko, M. T., Okuno, E., and Schwarcz, R. (2007) Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain, Journal of neurochemistry 102, 103-111.

7. Han, Q., Cai, T., Tagle, D. A., Robinson, H., and Li, J. (2008) Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II, Bioscience reports 28, 205-215.

8. Kocki, T., Luchowski, P., Luchowska, E., Wielosz, M., Turski, W. A., and Urbanska, E. M.

(2003) L-cysteine sulphinate, endogenous sulphur-containing amino acid, inhibits rat brain kynurenic acid production via selective interference with kynurenine aminotransferase II, Neurosci. Lett. 346, 97-100.

9. Han, Q., and Li, J. (2004) Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production, FEBS letters 577, 381-385.

10. Han, Q., Li, J., and Li, J. (2004) pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I, European journal of biochemistry / FEBS 271, 4804-4814.

11. Cuenod, M., Grandes, P., Zangerle, L., Streit, P., and Do, K. Q. (1993) Sulphur-containing excitatory amino acids in intercellular communication, Biochemical Society transactions 21, 72-77.

12. Kilpatrick, I. C., and Mozley, L. S. (1986) An initial analysis of the regional distribution of excitatory sulphur-containing amino acids in the rat brain, Neuroscience letters 72, 189-193.

13. Washko, M. E., and Rice, E. W. (1961) Determination of glucose by an improved enzymatic procedure, Clinical chemistry 7, 542-545.

14. Barnes, A. R., and Sugden, J. K. (1990) Comparison of colourimetric methods for ammonia determination, Pharm Acta Helv 65, 258-261.

15. Cook, P. F., and Cleland, W. W. (2007) Enzyme Kinetics and Mechanism, Taylor and Francis Group, LLC, New York.

16. Parsons, B., and Rainbow, T. C. (1984) Localization of cysteine sulfinic acid uptake sites in rat brain by quantitative autoradiography, Brain Res. 294, 193-197.

17. Weinstein, C. L., Haschemeyer, R. H., and Griffith, O. W. (1988) In vivo studies of cysteine metabolism. Use of D-cysteinesulfinate, a novel cysteinesulfinate decarboxylase inhibitor, to probe taurine and pyruvate synthesis, J. Biol. Chem. 263, 16568-16579.

18. Donini, S., Ferrari, M., Fedeli, C., Faini, M., Lamberto, I., Marletta, A. S., Mellini, L., Panini, M., Percudani, R., Pollegioni, L., Caldinelli, L., Petrucco, S., and Peracchi, A. (2009) Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism, Biochem. J. 422, 265-272.

19. Longenecker, J. B., and Snell, E. E. (1955) A possible mechanism for kynureninase action, J. Biol. Chem. 213, 229-235.

20. Dearden, J. C., and Forbes, W. F. (1958) LIGHT ABSORPTION STUDIES. PART XII.

ULTRAVIOLET ABSORPTION SPECTRA OF BENZALDEHYDES, Canadian Journal of Chemistry 36, 1362-1370.

21. Kim, H., Ikegami, K., Nakaoka, M., Yagi, M., Shibata, H., and Sawa, Y. (2003) Characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum, Biosci Biotechnol Biochem 67, 490-498.

22. Yagi, T., Kagamiyama, H., and Nozaki, M. (1979) Cysteine sulfinate transamination activity of aspartate aminotransferases, Biochem. Biophys. Res. Commun. 90, 447-452.

23. Graber, R., Kasper, P., Malashkevich, V. N., Strop, P., Gehring, H., Jansonius, J. N., and Christen, P. (1999) Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation, J. Biol. Chem. 274, 31203-31208.

24. Ghosh, N. K., and Fishman, W. H. (1966) On the mechanism of inhibition of intestinal alkaline phosphatase by L-phenylalanine. I. Kinetic studies, The Journal of biological chemistry 241, 2516-2522.

25. Nahorski, S. R., Ragan, C. I., and Challiss, R. A. (1991) Lithium and the phosphoinositide cycle: an example of uncompetitive inhibition and its pharmacological consequences, Trends in pharmacological sciences 12, 297-303.

26. Hoylaerts, M. F., Manes, T., and Millan, J. L. (1992) Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase, The Biochemical journal 286 ( Pt 1), 23-30.

27. Boocock, M. R., and Coggins, J. R. (1983) Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate, FEBS letters 154, 127-133.

28. Pollack, S. J., Atack, J. R., Knowles, M. R., McAllister, G., Ragan, C. I., Baker, R., Fletcher, S. R., Iversen, L. L., and Broughton, H. B. (1994) Mechanism of inositol monophosphatase, the putative target of lithium therapy, Proceedings of the National Academy of Sciences of the United States of America 91, 5766-5770.

29. Fell, D. A., and Snell, K. (1988) Control analysis of mammalian serine biosynthesis.

Feedback inhibition on the final step, The Biochemical journal 256, 97-101.

30. Morino, Y., Kojima, H., and Tanase, S. (1979) Affinity labeling of alanine aminotransferase by 3-chloro-L-alanine, J. Biol. Chem. 254, 279-285.

31. Cooper, A. J., Bruschi, S. A., Iriarte, A., and Martinez-Carrion, M. (2002) Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate beta-lyase reactions, Biochem.

J. 368, 253-261.

Documenti correlati